This proposed program can be considered as a competitive renewal of grant R01AI083754, titled Development of A Novel Strategy to Produce Antibacterial Glycoconjugate Vaccines, funded under ARRA from 7/30/2009 - 7/29/2011. The proposed work is aiming at solving an unmet biomedical need in the development of vaccines. Development of antibacterial vaccines provides an attractive approach for fighting bacterial diseases. Surface-located Polysaccharides (PSs) of bacteria have great potentials to be used as vaccines for preventing bacterial infections. Although traditional chemical conjugation of polysaccharides with carrier proteins to make PS-protein conjugate vaccines has resulted in several highly successful glycoconjugate vaccines for the clinical use, it still suffers variable batch-to-batch composition, difficult quality control, inconsistent potency and high production cost. The fatal problem is that such approach does not produce a structurally well defined, pure chemical entity, which can be linked to its immunological activity in subsequent structural- activity relationship (SAR) investigation, as it is routinely done in modern drug discovery programs for small molecules. The discovery and further development of bacterial protein N- glycosylation system have provided a novel approach to solve this biomedical problem. Oligosaccharyltransferase (PglB) from Campylobacter jejuni, which was first discovered by Markus Aebi in 2002, later developed by GlycoVaxyn LLC, was found to be able to transfer a variety of PS (from different bacteria) from its diphospho-undecaprenyl forms to the Asn of a consensus sequence of the target protein in the periplasm. Such synthetic biology approach fits well with our long-term efforts on studying the biosynthesis of microbial polysaccharides. Thus we have been developing this novel approach under ARRA R01AI083754. Our efforts resulted in a recent huge technique breakthrough after we worked out a facile method to clone any 20 to 30 kb polysaccharide biosynthesis gene clusters into an expression vector. Now structurally well-defined polysaccharide-protein bioconjugate can be produced by one-shot fermentation of recombinant E. coli K12 strain (incorporated with O-antigen gene cluster, pglB and carry protein gene acrA, each in one vector). For example, we recently produced 4.5 mg fully purified E. coli O157 O-antigen polysaccharide conjugated AcrA protein from simple E. coli fermentation. Such a bioconjugate already offers good possibility to be used to induce antibodies in cows or cattle to kill an inoculum of E. coli O157, since the vaccines can be produced in large scale economically. Therefore, the program aims to produce a series of PS-protein bioconjugates and variants, as well as study the SAR of several important bioconjugate vaccines. Aim 1: Production of PS-protein bioconjugates: Two classes of bacterial infections will be attached. The first includes E. coli O157, O104 and Shigella sonnei; the second includes Staphylococcus aureus. Aim 2: Production of PS-protein variants Our bacterial protein N-glycosylation platform allow us to change the length of polysaccharides, the length of the carrier proteins, the density of PS on the carrier protein and fusion of any immunologically active peptide or protein to the carrier protein. Such PS-protein variants will be available for the first time for SAR investigatio. Aim 3: Immunological studies on PS-protein bioconjugates Collaborating with immunologist colleagues at GSU (many more national and international collaborators as the program unfolds), the immunological activities of PS-protein bioconjugates will be investigated with the goal of finding better protective vaccines than current conventional approaches can offer.